The problems related to a generally low oxidation stability is a well known major obstacle in respect to the activity of detergent enzymes. Due to the presence of bleach active ingredients, the detergent enzymes have to perform their enzymatic action in an oxidative environment, with a consequent loss of activity.
Various solutions have been proposed to the problem, but hitherto oxidation stable detergent enzymes have not been available.
The currently used detergent enzymes have been found by isolating the enzymes from nature and testing them in detergent formulations. Some detergent enzymes have been artificially modified by deletions or substitutions of amino acids within their molecule, in order to achieve novel detergent enzymes with altered chemical and enzymatic properties. Techniques as random and site-directed mutagenesis have been applied from knowledge of the physical and chemical properties of the enzymes, and accordingly these techniques have mostly been applied to proteases.
Especially site-directed mutagenesis of the subtilisin genes has attracted much attention, and various mutations are described in patent publications (see e.g. EP 130,756; EP 214,435; EP 303,761; EP 260,105; WO 87/04461; W087/05050).
A subtilisin is a serine protease produced by Gram-positive bacteria or fungi. A wide variety of subtilisins have been identified, and in several cases the amino acid sequence has been determined (WO 89/06279 and WO 91/00345). In position 222 these wildtype subtilisins hold a methionyl residue, and this methionyl residue is identified as being responsible for the lability of the enzyme towards oxidative agents (J. Biol. Chem., 244, 5333-5338 (1969)).
Substitution of the residue at position 222 with any other of the 19 essential amino acid residues has been carried out, and the mutants obtained were investigated in respect to relative activity (J. Biol. Chem., 260, 6518-6521 (1985)). Only the Cys-mutant displayed a relative activity in magnitude of the wildtype (56%), but similar to the wildtype this Cys-mutant was unstable to oxidative agents, and a "half life" in the order of 12 minutes in 1 M H.sub.2 O.sub.2 was found (the wildtype-subtilisins do not contain cystein residues). Among the most oxidation stable mutants the Ala-mutant displayed the highest enzymatic activity (11%), and it did not lose its activity even after 15 minutes in 1 M H.sub.2 O.sub.2.
For scientific use it has been demonstrated that site-directed mutagenesis can be combined with chemical modifications in order to achieve enzyme derivatives with altered properties. Thus, a chemical modification of a cysteinyl residue introduced in the binding site of carboxy-peptidase-Y has proved to give enzyme derivatives that are more effective in deamidations of peptide amides and peptide synthesis, respectively (Bech, L.M. & Breddam, K.; Carlsberg Res. Commun., 53, 389-393 (1988)).
Therefore, it is an object of the present invention to provide novel chemically altered detergent enzymes, exhibiting improved oxidation resistance, and at the same time substantially retaining their proteolytic activity in respect of their wash performance. Further, it is an object of the present invention to establish a novel process for stabilizing detergent enzymes against oxidation.